Mortalin (mortalin 2) is a protein of 679 amino acids and having a molecular weight of 73,913 daltons. Its proprotein has a mitochondrial transfer signal peptide of 46 amino acids. Mortalin is a member of the Hsp70 family proteins, and it is a non-thermoresponsive protein. Mortalin has very high homology with Escherichia coli DnaK, yeast SSC1p, and Hsp70 family proteins such as Hsp70, which is constantly expressed in rat cytoplasmic fractions, Hsc70, and BiP, which is an isoform existing in rat endoplasmic reticula. Regarding mortalin, first, a mortalin 1 (mot-1) gene has been isolated from cytoplasmic fractions of normal mouse-derived fibroblasts (Wadhwa, R., Kaul, S. C., Ikawa, Y., and Sugimoto, Y. (1993) J Biol Chem 268, 6615-6621). Next, through immunological cloning of the cDNA of mouse immortalized cells and comparison with a sequence isolated from normal cells, the presence of a mortalin 2 gene (mot-2) encoding a protein differing by only 2 amino acid residues at the carboxyl terminus has been revealed (Wadhwa, R., Kaul, S. C., Sugimoto, Y., and Mitsui, Y. (1993) J BiolChem 268, 22239-22242). Mortalin 1 (mot-1) is present in normal cells, but mortalin 2 (mot-2) is present in immortalized cells. As revealed by nude mouse assay, whereas the expression of mortalin 1 (mot-1) causes a cellular-senescence-like phenotype, the overexpression of mortalin 2 (mot-2) causes malignant mutation. These indicates that mot-1 and mot2 exert biological activities that contrast one another (Wadhwa, R., Shyichi, T., Robert, M., Yoshida, A., Reddel, R. R., Nomura, H., Mitsui, Y., and Kaul, S. C. (1998) J Biol Chem 273, 29586-29591). Human mortalin has homology as high as 95% with mouse mortalin at the protein level and is also referred to as human mortalin 2 (hmot-2) because of the presence of only one type that exerts functions and properties similar to those of mortalin 2 (Kaul, S. C., Duncan, E. L., Englezou, A., Takano, S., Reddel, R. R., Mitsui, Y., and Wadhwa, R. (1998) Oncogene 17, 907-911). In the present invention, unless particularly specified, mouse mortalin 2 and human mortalin 2 are both simply referred to as mortalin or mortalin 2.
It has been suggested that mortalin 2 binds to various molecules at various positions within cells via calcium-dependent self-phosphorylation and then is involved in wide-ranging functions including mitochondrial transport, intracellular transport, chaperonin functions, stress responses, tumorigenesis, and the like. In particular, it has been revealed that: mortalin 2 binds to p53, a tumor suppressor protein, so as to inactivate the transcriptional activity functions of p53 (Wadhwa, R., Shyichi, T., Robert, M., Yoshida, A., Reddel, R. R., Nomura, H., Mitsui, Y., and Kaul, S. C. (1998) J Biol Chem 273, 29586-29591); and mortalin 2 cooperates with telomerase to immortalize human foreskin fibroblasts (Kaul, S. C., Yaguchi, T., Taira, K., Reddel, R. R., and Wadhwa, R. (2002) ECR submitted), for example. Hence, essential involvement of mortalin 2 in oncogenesis has been revealed (Kaul, S. C., Taira, K., Pereira-Smith, O. M., and Wadhwa, R. (2002) Exp Gerontol 37, 1157-1164; Wadhwa, R., Takano, S., Kaur, K., Deocaris, C. C., Pereira-Smith, O. M., Reddel, R. R., and Kaul, S. C. (2006) Int J Cancer 118, 2973-2980; Deocaris, C. C., Kaul, S. C., and Wadhwa, R. (2006) Cell Stress Chaperones 11, 116-128; Dundas, S. R., Lawrie, L. C., Rooney, P. H., and Murray, G. I. (2005) J Pathol 205, 74-81; Shin, B. K., Wang, H., Yim, A. M., Le Naour, F., Brichory, F., Jang, J. H., Zhao, R., Purays, E., Tra, J., Michael, C. W., Misek, D. E., and Hanash, S. M. (2003) J Biol Chem 278, 7607-7616; Pizzatti, L., Sa, L. A., de Souza, J. M., Bisch, P. M., and Abdelhay, E. (2006) Biochim Biophys Acta 1764, 929-942; Walker, C., Bottger, S., and Low, B. (2006) Am J Pathol 168, 1526-1530; and International Patent Publication WO2006/022344 A1 and JP Patent Publication (Kokai) No. 2006-89471 A. It has also been shown that the possibility exists for a molecule such as an anti-mortalin antibody to bind to mortalin, so as to suppress the effects and functions of mortalin, can be used as an anticancer agent (Walker, C., Bottger, S., and Low, B. (2006) Am J Pathol 168, 1526-1530; Wadhwa, R., Sugihara, T., Yoshida, A., Nomura, H., Reddel, R. R., Simpson, R., Maruta, H., and Kaul, S. C. (2000) Cancer Res 60, 6818-6821; Wadhwa, R., Ando, H., Kawasaki, H., Taira, K., and Kaul, S. C. (2003) EMBO Rep 4, 595-601; Deocaris, C. C., Widodo, N., Shrestha, B. G, Kaur, K., Ohtaka, M., Yamasaki, K., Kaul, S. C., and Wadhwa, R. (2007) Cancer Lett (in press)).
The present inventors have previously examined whether mortalin can be an effective target for cancer treatment, in addition to examination of the relationship between an increased expression level of mortalin and oncogenesis. Thus, they have applied for a patent relating to an anti-mortalin antibody having a internalizing function into cancer cells, a pharmaceutical composition for cancer treatment using the antibody, a drug carrier, and the like (International Patent Publication WO2006/022344 A1, JP Patent Publication (Kokai) No. 2006-89471 A). Such anti-mortalin antibody having the internalizing function into cancer cells can be used itself not only as an antibody drug, but also as a drug carrier that delivers immunotoxins or the like to tumor cells.
In the course of examining them, the present inventors have observed that not all antibodies specifically recognizing mortalin have such internalizing function into cancer cells. Thy have also observed the presence of antibodies capable of specifically recognizing mortalin, but incapable of being internalized into cancer cells. However, essential differences in terms of amino acid sequences or structures between antibodies having a internalizing function into cancer cells and antibodies not having such function have not been elucidated. Hence, the kind of region in the full-length antibody that is involved in the internalization mechanism has remained completely unknown.
Drugs preferred herein are humanized antibodies with low immunogenicity that causes low adverse reaction in normal cells or drugs characterized in that only a region that is as short as possible can be administered. Accordingly, in addition to elucidation of the internalization mechanism of a mortalin antibody, elucidation of a region involved in internalization has been desired.
Also, as a first step for internalization of an anti-mortalin antibody within cancer cells, first, interaction of the antibody with mortalin is thought to be essential. Hence, elucidation of a site on mortalin involved in the interaction with an anti-mortalin antibody, and particularly, elucidation of recognition regions recognized by antibodies having the internalizing function and antibodies not having such function, have also been desired.
Furthermore, if an epitope sequence to be recognized by an antibody having the internalizing function can be determined, the epitope is expressed on cancer cell surfaces using the nucleic acid molecule encoding the epitope, so that internalization of the anti-mortalin antibody can be accelerated. Therefore, in particular, the sequencing of an epitope of an antibody having the internalizing function has been strongly desired.
All descriptions of these cited documents are incorporated into the specification of the present application.